Structural and functional analysis of LIM domain-dependent recruitment of paxillin to focal adhesions

Abstract The LIM domain-dependent localization of the adapter protein paxillin to focal adhesions (FAs) is not mechanistically understood. Here, by combining molecular biology with photoactivation and FA-isolation experiments, we demonstrate a specific contribution of each LIM domain and reveal the existence of multiple paxillin docking sites in the FA-complex. Mutation of β3 integrin at a putative paxillin binding site leads to rapid inward sliding of FAs, correlating with enhanced paxillin dissociation rates. Mechanical coupling of paxillin to integrins or the plasma membrane arrests the FAs sliding, thereby disclosing an essential structural function of the LIM-array for the maturation of integrin/talin clusters. Moreover, via bimolecular fluorescence complementation, we determine a precise spatial orientation of the paxillin LIM domains, juxtaposing the positively charged LIM4 to the plasma membrane and the extremity of the β3 integrin tail, providing structural insights into the molecular organization of FAs.